Adeno-associated virus type 2 (AAV2) is a naturally defective human parvovirus that is being developed as a gene therapy vector. In dividing cells AAV2 DNA persists by integration into the host chromosomes. AAV2 is unique among mammalian viruses in its ability to preferentially integrate into a particular locus within human chromosome 19, designated AAVS1 (also known as Mbs 85). The AAV2 Rep68 and Rep78 proteins mediate this preferential integration. Recent data suggest that Rep68 and Rep78 can mediate preferential integration of any linear viral DNA. To test this hypothesis, we co-infected wild-type AAV2 with a lentiviral vector and were able to detect integration of the lentiviral vector into AAVS1, using a PCR-based assay. In parallel, we have constructed hybrid lentivirus vectors containing the AAV2 rep gene. PCR data indicates that these hybrid lentivirus vectors also target AAVS1. Junction formation was confirmed by sequence analysis of PCR products. Only the amino acid sequence shared between Rep68 and Rep78 is required for preferential integration. Our results were similar whether or not the vector contained a functional lentivirus integrase gene. The inclusion of the rep gene in lentivirus vectors may reduce the risks associated with insertional mutagenesis.